In recent years, in the field of blood transfusion, a leukocyte-free blood transfusion in which leukocytes are removed from a blood product before transfusion is increasingly employed. This is because it has become apparent that side effects of transfusion, such as headache, nausea, chills and non-hemolytic feverish reaction, and side effects more serious to a recipient, such as allosensitization, post-transfusion GVHD (graft versus host disease) and viral infection, are mainly caused by leukocytes contained in a blood product employed in transfusion.
It is known that the number of leukocytes injected into a recipient at one transfusion must be limited to about 100,000,000 or less in order to avoid relatively slight side effects, such as headache, nausea, chills and fever. For meeting this requirement, leukocytes must be removed from a blood product to a level of 10.sup.-1 to 10.sup.-2 or less in terms of a leukocyte residual ratio. With respect to allosensitization, it now attracts the greatest attention in the art of blood transfusion, and it is one of the side effects, prevention of which is most desired. For preventing this serious side effect, it is believed that the number of leukocytes injected into a recipient at one transfusion must be limited to 5,000,000 or less, preferably 1,000,000 or less. For meeting this requirement, leukocytes must be removed from a blood product to a level of 10.sup.-4 or less in terms of a leukocyte residual ratio. With respect to post-transfusion GVHD and viral infection, no generally accepted standards for leukocyte-removal have been established. However, it is expected that infection with a virus, which is believed to exist only in leukocytes, such as cytomegalo virus, adult T cell leukemia virus and post-transfusion GVHD, could be prevented by removing leukocytes to a level of 10.sup.-4 to 10.sup.-6 or less in terms of a leukocyte residual ratio. Further, it is also expected that the probability of infection with a virus, which is believed to exist in both leukocytes and plasma, such as HIV, can be decreased by removing leukocytes.
The methods for removing leukocytes from a blood product can generally be classified into two methods. One is a method in which leukocytes are separated by a centrifuge, taking advantage of a specific gravity difference therebetween. The other is a filtering method in which leukocytes are removed by a filter comprising a fiber material or a spongy structure as a filter medium. In particular, a filtering method in which leukocytes are adsorption-removed by a non-woven fabric is widely employed due to the advantages of high capability to remove leukocytes, ease in handling and low cost.
Most of the conventional leukocyte-removing filters comprising a non-woven fabric are composed of two functionally different filter elements, i.e., a prefilter for removing aggregates, which has an average fiber diameter of from about 3 to 30 .mu.m and a relatively large pore size, and a main filter as an essential element for removing leukocytes, which is comprised of fibers having an average diameter of from about 1.7 to 3 .mu.m. With respect to the above-mentioned prefilter, it is preferably comprised of a plurality of layers in which the average fiber diameters and the pore sizes of the layers are decreased in the direction from a blood inlet toward a blood outlet (Japanese Patent Application Publication Specification No. 2-13588 and WO 89/03717). Aggregates are formed by aggregation of denatured blood components comprising fibrinogen, fibrin, denatured protein, nucleic acid and/or fat globule, or by aggregation of cellular components, such as leukocytes and platelets. The aggregates are highly sticky, and their sizes have a very broad distribution, i.e., from several microns to 100 .mu.m, sometimes exceeding 1 mm. Accordingly, as in the case of separating particles with sieves, it is requisite to first capture and remove large aggregates with a filter having a large pore size, and then to remove smaller aggregates by the use of filters having stepwise decreasing pore sizes. Some of the leukocytes may rather secondarily be captured by a prefilter at its layer having the smallest pore size, which layer is to be utilized to remove small aggregates. However, the proportion of such captured leukocytes is extremely small. Essentially, the removal of leukocytes must be effected by the use of a main filter as described below.
Leukocytes as the principal matter to be removed according to the present invention have a diameter of from 5 to 20 .mu.m, and their sizes are much more uniform than those of aggregates. It is believed that the removal of leukocytes by a filter is due to the adsorption thereof onto the fibers contained in the filter. The present inventors previously found that the concentration of leukocytes passing through a fiber laminate decreases in exponential relationship with the thickness of the fiber laminate (Japanese Patent Application No. 1-296269). This suggests that leukocytes are adsorbed with a certain probability onto fibers at every contact of leukocytes with fibers around their crossover points during the flow of leukocytes through the fiber laminate in the direction of the thickness of the fiber laminate, and hence supports the above belief that the removal of leukocytes by a filter is due to the adsorption thereof onto the fibers.
The terminology "crossover point" used herein is defined below. That is, in a filter element comprised of a large number of fibers, the "crossover point" refers to points where at least two fibers are engaged with each other in a crossing relationship, points where at least two fibers are engaged with each other in a grade separation relationship with a minor spacing therebetween which is smaller than the diameter of each leukocyte, and points where at least two fibers are engaged with each other in an adjacent relationship with an inter-fiber spacing therebetween which is smaller than the diameter of each leukocyte.
Accordingly, the conventional investigations on a main filter as a member of a filter for removing leukocytes have been focused on increasing the above adsorption probability, i.e., decreasing an average fiber diameter, increasing a packing density, and the like.
On the other hand, in H. Prins; use of microfiber nonwovens in blood filtration, Session Applications 1-Filtration & Separation, Index 90 Congress, N.P.B.I., The Netherlands, it is described that among leukocytes, granulocytes are removed by adhesion and lymphocytes are removed by sieving mechanism so that it is necessary to employ functionally different filter elements, one being adapted for granulocytes and the other being adapted for lymphocytes. Further, it is described that a filter element having an average fiber diameter of 5 .mu.m was used for the removal of granulocytes while a filter element having an average fiber diameter of 2.5 .mu.m was used for the removal of lymphocytes. However, the filter disclosed in this reference is of a large size, and no contemplation appears in the reference with respect to improvement of the performance of the filter, e.g., removal of leukocytes to a level of 10.sup.-4 or less in terms of a final leukocyte residual ratio. Actually, the filter element having an average fiber diameter of 5 .mu.m corresponds to the above-mentioned prefilter. Although this filter element removes a portion of leukocytes, the primary function thereof is to remove small aggregates as described in the reference as well. On the other hand, the filter element having an average fiber diameter of 2.5 .mu.m which is used for removing lymphocytes, also removes a large proportion of granulocytes, and this filter element corresponds to the above-mentioned conventional main filter. Accordingly, no problem is posed even if both granulocytes and lymphocytes are removed by a filter element having an average fiber diameter of 2.5 .mu.m without the removing of granulocytes by a filter element having an average fiber diameter of 5 .mu.m. That is, the filter element having an average fiber diameter of 5 .mu.m is not considered as an element which is indispensable for the removal of leukocytes. Actually, in the reference, no contemplation is made with respect to the necessity to remove granulocytes by means of a filter element having an average fiber diameter of 5 .mu.m prior to the use of a filter element having an average fiber diameter of 2.5 .mu.m.
Moreover, the filter element having an average fiber diameter of 2.5 .mu.m corresponds to the conventional main filter as mentioned above, and hence a high leukocyte removal efficiency cannot be expected with respect to this type of filter element.
Illustratively stated, in the reference, there is neither intention to remove granulocytes by means of a filter element having an average fiber diameter of 5 .mu.m (which can be regarded as a prefilter in the reference) prior to the filtration by the use of a filter element having an average fiber diameter of 2.5 .mu.m (which can be regarded as a main filter in the reference), nor recognition of the necessity of the same.
Recently, in accordance with wider recognition of the importance of leukocyte-free blood transfusion, a filter for removing leukocytes which exhibits an improved leukocyte removal efficiency and has a smaller size and a smaller internal space volume, has been demanded in the art. Usually, a blood product remaining inside a filter after filtration operation to remove leukocytes is discarded together with the filter. Therefore, to minimize the waste of blood product, a filter having a small internal space volume is demanded. The terminology "internal space volume" used herein means the volume of the whole space inside a filter in which a filter element is placed, the whole space including the void portion of the filter element. With respect to the capability of removing leukocytes, even the most effective filter known in the art can remove leukocytes to a level of a leukocyte residual ratio of about 10.sup.-3 at the best, and further it cannot avoid the above-mentioned serious side effects. Generally, it is desired that the proportion of blood product discarded with a filter be limited to a level as low as 10-15% or less. For meeting this requirement, it is desired that the filter be of a small size such that it has an internal space volume as small as 35 ml or less per unit of whole blood or red cell product or as small as 20 ml or less per 5 units of platelet product. However, the conventional filters cannot simultaneously realize both high leukocyte removal efficiency and satisfactorily small internal space volume.
The measures for improving leukocyte removal efficiency which can readily be conceived by persons skilled in the art would be to increase the amount of main filter or to employ a filter element having a smaller average fiber diameter. For increasing the amount of main filter while keeping the internal space volume thereof at the above-mentioned value or less, it is necessary to increase the packing density of main filter in a container so as to suppress the internal space volume. Generally, the upper limit of the packing density is about 0.4 g/cm.sup.3. At a higher packing density, it is difficult to accomplish the packing in a container due to the repulsive force of non-woven fabric. To avoid this difficulty, heat pressing or the like may be performed for the non-woven fabric. However, this results in a collapse of the non-woven fabric into a film, which can no longer function as a filter. Therefore, for improving leukocyte removal efficiency, it is necessary to adopt a method in which the amount of main filter is increased while keeping the packing density at 0.4 g/cm.sup.3 or less, or a method in which a main filter having a smaller average fiber diameter is used (as a result of the studies of the present inventors, it has been found that in order to attain a leukocyte residual ratio of 10.sup.-4 or less while keeping the packing density at a value within the above-mentioned range, the average fiber diameter of non-woven fabric must be 1.6 .mu.m or less).
In either method, however, there has been a problem that the improvement of the capability of leukocyte removal is inevitably accompanied by an increase of a pressure loss in a main filter region during the passage of a blood product so that filtering speed is drastically lowered before the completion of filtration of a predetermined amount of blood.
As mentioned hereinbefore, according to the finding of the present inventors, the concentration of leukocytes passing a fiber laminate is decreased in an exponential relationship with the thickness of the fiber laminate in the passage of a blood product through the fiber laminate. Further, it has been found that the smaller the average fiber diameter and the higher the packing density, the greater the degree of this decrease. Accordingly, an increase in the capability of leukocyte removal with an internal space volume limited to within the above-mentioned range may be performed by employing fibers having a small average diameter or increasing a packing density in a main filter. However, these measures are inevitably accompanied by serious problems, as mentioned hereinbefore. That is, when these measures are actually adopted in order to improve a leukocyte residual ratio to 10.sup.-4 or less, the leukocyte-removing ability is improved but a pressure loss is increased in a main filter region during the passage of a blood product with the improvement of the capability of the main filter to remove leukocytes, so that filtering speed is drastically lowered before the completion of filtration of a predetermined amount of blood. With a view toward elucidating the reason for the problems, the present inventors have made intensive studies with respect to the factors increasing the pressure loss of a filter. As a result, it has unexpectedly been found that the major reason for the problems is a clogging of the filter with leukocytes.
Before the present invention, it has generally been believed that the pressure loss of a filter is essentially attributed to a viscous resistance of blood passing through the filter, and that the voids of the filter through which blood can flow are clogged with aggregates and thus an increase in pressure loss accompanying blood filtration occurs due to an increase in linear velocity of blood in the remaining unclogged portions. Further, it has been known since the time a flocculent fiber mass was used in a leukocyte-removing filter that blood flow rate is drastically lowered when fibers having a small average fiber diameter are employed and that the same occurs when packing density is increased. In such a case, however, blood flow rate is low from the initial stage of blood filtration. This is a phenomenon which is clearly different from the problem of flow rate being rapidly lowered from the middle of blood filtration as currently encountered by the present inventors. That is, the problem is attributed to a sharp increase in viscous resistance of blood which is caused by effecting a diameter minimization and a packing density increase for a flocculent fiber mass. This problem has been solved by the use of a non-woven fabric as a filter material, as disclosed in Japanese Patent Application Publication Specification No. 2-13587.
In fact, the degree of viscous resistance of blood has never become a problem with respect to the conventional leukocyte-removing filters comprised of a non-woven fabric, which filters can remove leukocytes to a level of a leukocyte residual ratio of about 10.sup.-3 or so. Problems relating to pressure loss or flow rate, if any, have been concerned with clogging of the filter with aggregates which increased during the storage of blood, especially in the case of filtration of old blood which has been stored for a prolonged period of time, which clogging causes a pressure loss increase and a filtration speed lowering from the middle of blood filtration. These problems have also been solved by employing a prefilter for removal of aggregates as disclosed in Japanese Patent Application Publication Specification No. 2-13588, and such filters have conventionally been widely utilized in the art.